Serum levels of testosterone are reduced as humans and rodents age. Previous studies of the rat have provided correlative evidence suggesting that age-related increases in oxidative stress and/or deficiencies in the antioxidant defense system may be involved in the reduced ability of aged Leydig cells to produce testosterone. Transgenic and knockout animals represent powerful tools by which to relate candidate genes and their protein products to physiologic outcomes, and thus to examine cause-effect relationships. The major goal of the studies that are proposed herein is to utilize this approach to gain the information needed to begin to test cause-effect relationships between free radical production and the steroidogenic function of aging Leydig cells. The first aim of the proposal is to describe parameters of Leydig cell aging in the mouse by testing the hypotheses that, with aging, testosterone production by Leydig cells of C57BL/6 mice is reduced, mitochondrial superoxide production increases, and there are deficits in the ROS scavenging system (SOD, glutathione peroxidase, catalase) of aged cells. The second aim will focus on transgenic mice that overexpress Gpx4 and on Gpx4 knockdown/knockout mice to test the hypothesis that the overexpression of protective proteins results in suppression or delay in age-related steroidogenic decline that is characteristic of aging Leydig cells, and that deficiencies in the antioxidant defense system result in acceleration or exacerbation of the decline. The successful completion of this research will allow the free radical theory of aging to begin to be tested in aging Leydig cells, an exceptionally well defined system. If successful, the results will begin to move analyses of the "free radical theory of aging" from compelling, though correlative, analyses to ascribing cause and effect. Age-related decline in testosterone in men has relevance to important quality of life issues such as increased osteoporosis, reduced cognition and reduced libido in the aging male population. Studying how testosterone decreases, and what might be done to prevent this from occurring, will provide new insights into how Leydig cells cope with stressors that are present (or increase) with aging, shed light on the underlying molecular basis for age-related functional changes in the Leydig cells, and provide clues as to how to prevent or reverse these changes. [unreadable] [unreadable] [unreadable]